One of the most common types of assays used as a rapid point of care test to detect a particular analyte in a biological sample is a lateral flow strip-based assay. Such assays typically contain a binding partner for the analyte of interest coupled to a detectable label (i.e. labeled conjugates) and a porous membrane on which a capture protein (e.g. antibody or antigen) capable of binding the analyte of interest is immobilized. Labeled conjugates that are commonly used in these types of assays are antibodies or antigens coupled to gold nanoparticles or colored latex particles. An analyte present in the sample will bind to the labeled conjugate to form a complex. The complex continues to migrate through the porous membrane to the region where the capture protein is immobilized at which point the complex of analyte and labeled conjugate will bind to the capture protein. The presence of the analyte is then determined by detecting the labeled conjugate in the capture region of the porous membrane (e.g. by a color change of the capture line).
Although lateral flow strip-based assays have proven useful for rapid detection of some analytes in the clinical setting, such assays are inherently limited by their sensitivity due to the occurrence of a single binding event. Amplification of signals can be achieved, for example, by employing an enzyme as the detectable label. Lateral flow assays that employ enzyme label conjugates have been developed. However, these assays require multiple steps to wash the enzyme label conjugate from the capture region and to subsequently deliver the enzyme substrate. Amplification can also be effected in assays that employ nanoparticles as the detection label by intensifying the signal with silver particles (e.g. application of silver nitrate) or by deposition of reaction products from alkaline phosphatase, peroxidase, or β-galactosidase reactions. Similar to amplification in assays using enzyme label conjugates, amplification in nanoparticle-based assays also requires several steps to wash the unreacted conjugate from the capture region and apply the intensification solution.
Thus, there is a need in the art to develop one-step, e.g. rapid lateral flow assays with built-in amplification to provide sensitivity for the detection of analytes in samples, particularly biological samples.